Method for the High Level Expression of Active Lymphotoxin-Beta Receptor Immunoglobulin Chimeric Proteins and Their Purification

ABSTRACT

Methods for high level expression of active lymphotoxin-β receptor immunoglobulin chimeric proteins and their purification.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/542,614, filed Aug. 17, 2009 which is a divisional of U.S.application Ser. No. 09/767,370, filed Jan. 23, 2001 which is acontinuation of International Application No. PCT/US99/29873, filed Dec.16, 1999, which claims the benefit of U.S. Provisional Application No.60/112,752, filed Dec. 17, 1998. The entire contents of each of theseapplications are hereby incorporated by reference herein.

BACKGROUND

The TNF family consists of pairs of ligands and their specific receptorsreferred to as TNF family ligands and TNF family receptors (Bazzoni andBeutler, 1996; Aggarwal and Natarajan, 1996). The ligands such as TNFare typically found as membrane bound forms on cell surfaces, or in somecases they are selectively cleaved from the cell surface and secreted.The ligands bind to specific receptors and the binding event serves toaggregate two or more receptors. The intracellular domains of thesereceptors can in some way sense this change and communicate thisinformation into the cell via a signal transduction mechanism. Thefamily is involved in the regulation of the immune system and possiblyother non-immunological systems. The regulation is often at a “masterswitch” level such that TNF family signaling can result in a largenumber of subsequent events best typified by TNF. TNF can initiate thegeneral protective inflammatory response of an organism to foreigninvasion which involves the altered display of adhesion moleculesinvolved in cell trafficking, chemokine production to drive specificcells into specific compartments and the priming of various effectorcells. As such, the regulation of these pathways has clinical potential.

The TNF receptor family is a collection of related proteins thatgenerally consist of an extracellular domain, a transmembrane domain andan intracellular signaling domain. The extracellular domain is builtfrom 2-6 copies of a tightly disulphide-bonded domain and is recognizedon the basis of the unique arrangement of cysteine residues (Banner etal, 1993). Each receptor binds to a corresponding ligand(s) although oneligand may share several receptors. In some cases, it is clear thatsoluble forms of the receptors lacking the transmembrane region and/orintracellular domain exist naturally. In nature, truncated versions ofthese receptors may have direct biological regulatory roles. An exampleof this process is provided by the osteoprotegerin system.Osteoprotegerin is a secreted TNF family receptor that blocks signalingvia the RANK-L (also called TRANCE) and/or TRAIL to receptors thattrigger osteoclast activation. By blocking these receptors, most likelyRANK receptor, bone resorption is hindered and bone mass increases(Bucay et al, 1998). Clearly, viruses have used this tactic to inhibitTNF activity in their host organisms (Smith et al, 1994). Thesereceptors can signal a number of events including cell differentiation,cell death or cell survival signals. Cell death signaling often istriggered via relatively direct links to the cascade of caspaseproteases in the case of the Fas and TNF receptors.

The TNF receptors are powerful tools to elucidate biological pathwayssince they are easily converted to immunoglobulin fusion proteins whichhave long serum halflives. Dimeric soluble receptor forms may beinhibitors of events mediated by either natural secreted or surfacebound ligands. By binding to these ligands these fusion proteins preventthe ligand from interacting with cell associated receptors and inhibitthe associated signal. These receptor-Ig fusion proteins are useful inan experimental sense, and have also been successfully used clinically,for example TNF-R-Ig has been used to treat inflammatory bowel disease,rheumatoid arthritis and the acute clinical syndrome accompanying OKT3administration (Eason et al., 1996; Feldmann et al., 1997; van Dullemenet al., 1995). The manipulation of the many events mediated by signalingthrough the TNF family of receptors may have application in thetreatment of immune based diseases as well as the wide range of humandiseases that have pathological sequelae due to immune systeminvolvement. For example, a soluble form of a recently describedreceptor, osteoprotegerin, has been shown to block the loss of bone mass(Simmonet et al, 1997). Thus, the events controlled by TNF familyreceptor signaling are not necessarily limited to immune systemregulation. Antibodies to receptors can block ligand binding and thusalso have clinical application. Such antibodies are often verylong-lived and may have advantages over soluble receptor-Ig fusionproteins which have shorter half-lives in the blood.

While inhibition of the receptor-mediated pathway represents the mostexploited therapeutic application of these receptors, originally it wasthe activation of the TNF receptors that showed clinical promise(Aggarwal and Natarajan, 1996). Activation of the TNF receptors caninitiate cell death in the target cell and hence the application totumors was and still is attractive (Eggermont et al., 1996). Thereceptor can be activated either by administration of the ligand, i.e.the natural pathway or by administration of antibodies that cancrosslink the receptor. Antibodies may be advantageous for the treatmentof, for example, cancers, since the antibodies can persist in the bloodfor long periods as opposed to ligands, which generally have shortlifespans in the blood. Agonist antibodies are useful weapons in thetreatment of cancer since receptors may be expressed more selectively intumors or they may only signal cell death or differentiation in tumors.Likewise, many positive immunological events are mediated via the TNFfamily receptors, e.g. host inflammatory reactions, antibody productionetc. and therefore agonistic antibodies could have beneficial effects inother, non-oncological applications.

Paradoxically, the inhibition of a pathway may also have clinicalbenefit in the treatment of tumors. For example the Fas ligand isexpressed by some tumors and this expression can lead to the death ofFas positive lymphocytes, thus facilitating the ability of the tumor toevade the immune system. In this case, inhibition of the Fas systemcould then allow the immune system to react to the tumor in other waysnow that access is possible (Green and Ware, 1997).

One member of this receptor family, the lymphotoxin-beta receptor (LTβR)binds to surface lymphotoxin (LT) which is composed of a trimericcomplex of lymphotoxin alpha and beta chains (Crowe et al, 1994). Thisreceptor-ligand pair is involved in the development of the peripheralimmune system and the regulation of events in the lymph nodes and spleenin the mature immune system (Ware et al, 1995; Mackay et al, 1997;Rennert et al, 1996; Rennert et al, 1997; Chaplin and Fu, 1998). Alymphotoxin-β receptor-immunoglobulin fusion protein can be made betweenLTβR and IgG (LTβR-Ig) that blocks signaling between the surface LTligand and the receptor with consequences on the functional state offollicular dendritic cells (Mackay and Browning 1998). This blocking canfurthermore lead to diminished autoimmune disease in rodent models(Mackay et al, 1998, U.S. Ser. No. 08/505,606 filed Jul. 21, 1995 andU.S. Ser. No. 60/029,060 filed Oct. 26, 1996). A second member of thisreceptor family called HVEM for herpes virus entry mediator binds to aligand called Light (Mauri et al, 1998) as well as the herteromeric LTligand. The function of this receptor is currently unknown, but aHVEM-Ig fusion protein may be useful for the treatment of immunologicaldisease and this construct has been shown to affect in vitro immunefunction assays (Harrop, J. A., et al, 1998).

Despite the clinical advances of members of the TNF Family as discussedabove, there remains a need for a method of obtaining the desired yieldsof receptor Ig fusions suitable for use in a clinical setting. Forexample, the LTβR-Ig protein can come in two forms when expressed ineither monkey cos cells or in Chinese hamster ovary cells. One formbinds ligand with high affinity whereas the other does not. Therefore,there is a need for a method for producing higher yields of the formwhich binds with high affinity, while minimizing the presence of thelower affinity form.

SUMMARY OF THE INVENTION

The present invention relates to methods for the expression of highyields of the form of protein-Ig fusions having high affinity binding toits ligand, referred to herein as the “active” form, the form, byculturing hosts transformed with DNA encoding the desired fusions in aculture system at a low temperature thereby minimizing the amount ofmisfolded or misbridged protein forms. The invention in variousembodiments relates to methods of expressing high yields in mammalianexpression systems, by culturing transformed hosts at a temperature ofabout 27° C. to about 35° C. Preferably, mammalian hosts will becultured at a temperature of about 27° C. to about 32° C.

In yet other embodiments the invention relates to methods for theexpression of high yields of active fusion proteins by culturingtransformed hosts in a yeast expression system at low temperatures. Whenthe desired proteins are expressed in yeast, the preferred temperaturesare from about 10° C. to about 25° C., more preferably from about 15° C.to about 20° C.

In certain methods of the claimed invention, the protein-Ig fusioncomprises a member of the TNF receptor family such as a lymphotoxin-βreceptor or a fragment thereof. Alternatively, the claimed methods mayencompass the expression of a desired fusion protein in any expressionsystem at low temperatures, such as an insect or bacterial system.

In other embodiments, the claimed invention encompasses the activeprotein-Ig fusions that are obtained by the claimed methods, andpharmaceutical compositions comprising them. In yet other embodiments,the invention relates to methods of making pharmaceutical preparationscomprising culturing a host transformed with DNA encoding a desiredprotein-Ig fusion in a culture system having a low temperature of about27° C. to about 35° C., preferably about 27° C. to about 32° C., toexpress a high yield of active fusion proteins, recovering the activeprotein fusions from the culture system, and combining the active fusionproteins recovered with a pharmaceutically acceptable carrier. Inpreferred embodiments, the protein-Ig fusion comprises a lymphotoxin-βor a fragment thereof, or HVEM, or a fragment thereof.

In still other embodiments, the claimed invention relates topharmaceutical compositions obtained using the methods described.

The claimed invention in different embodiments relates to mammalianexpression systems, as well as other expression systems, such as yeast,bacterial systems, or insect systems.

In certain embodiments, the invention relates to methods for high levelsof expression of active fusion proteins in yeast, by culturing at lowtemperatures of about 10° C. to about 25° C., more preferably, about 15°C. to about 20° C. Active fusions obtained by this method of expressionin yeast, and pharmaceutical compositions comprising these activefusions are also encompassed. Methods of making pharmaceuticalpreparations are encompassed within the invention comprising activeprotein-Ig fusions comprising culturing a yeast cell tranformed with DNAencoding a desired fusion at a low temperature, preferably about 10° C.to about 25° C., more preferably about 15° C. to about 20° C. In themost preferred embodiments of all the compositions and methods of theinvention, the protein-Ig fusion comprises a lymphotoxin-β receptor,HVEM, or a fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic of the receptor-immunoglobulin chimeric protein. Theleft panel shows a cartoon of a typical IgG molecule, the Fc domains arefilled in black, the heavy chain variable domains are colored in grayand the light chains are left white. The middle panel contains aschematic drawing of the LTβR molecule including the intracellular (darkgray) and extracellular (light gray) domains. The right panel depicts aschematic drawing of the LTβR-Ig fusion protein.

FIG. 2: Conceptual schematic showing the likely defect in the deadLTβR-Ig form, although the actual misfolded or misbridged amino acidshave not been identified.

FIG. 3: SDS-PAGE analysis of human LTβR-Ig before and after treatmentwith PNGase F. Lanes 1 and 2 contain human LTβR-Ig purified from CHOcell culture supernatant grown at 37° C. using Protein A affinitychromatography. Lane 3 shows the same preparation after treatment withPNGase F to remove all N-linked oligosaccharides. Lane 1 is run undernonreducing conditions, lanes 2 and 3 are run under reducing conditions.The protein bands are visualized by staining the gel with CoomassieBrilliant Blue.

FIG. 4: A nonreduced SDS polyacrylamide gel analysis of the proteinflowing through an AGH1 affinity column lanes and the protein elutedwith pH 3.5 phosphate buffer. The protein before affinity purificationis shown in the lane on the right side of the gel. The protein bands arevisualized by staining the gel with Coomassie Brilliant Blue.

FIG. 5: Ability of the flow through (lower) and eluted (upper) fractionsfrom the AGH1 affinity column to bind to surface lymphotoxin. Meanfluorescence intensity values were derived from a FACS analysis asdescribed. Examples of the FACS profiles are shown on the right wherethe LTβR-Ig is compared to the binding of a control LFA-3-Ig protein.

FIG. 6: Analytical HIC chromatography of human LTβR-Ig using a POROSether column and a decreasing gradient of ammonium sulfate. Peak 1represents the lower, inactive form, peak 2 the larger, active form ofhuman LTβR-Ig. Fractions corresponding to peaks 1 and 2 respectivelywere pooled and analyzed on a 4-20% SDS-PAGE gel (inserted panel). Thelane labeled “1” contains the “inactive” material from peak 1, lane 2the “active” material from peak 2 of the HIC chromatogram. The startingmaterial is shown in lane “L”. The lane labeled “M” contains molecularweight markers.

FIG. 7: Preparative HIC (hydrophobic interaction chromatography)chromatography of human LTβR-Ig using a Pharmacia Source 15 PHE column.Protein A eluate containing human LTβR-Ig is adjusted with 5 M NaCl to afinal concentration of 1.5 M NaCl, 20 mM sodium phosphate, pH 7.0 andloaded onto the column. The column was washed with 5 volumes of 1.5 MNaCl, 20 mM sodium phosphate, pH 7.0 and eluted with 20 mM sodiumphosphate, pH 7.0. The X-axis represents the time in minutes, the Y-axisshows the absorbance at 280 nm. Shown in the figure insert is the imageof a nonreducing 4-20% SDS-PAGE gel stained with Coomassie BrilliantBlue of the flow through (FT) and elution (E) pools. The migrationpositions of the “live” and “dead” forms are indicated with arrows.

FIG. 8: SDS-PAGE/western blot analysis of human LTβR-Ig secreted fromCHO cells grown at different temperatures. The CHO cell supernatantsgrown at the indicated culture temperatures containing human LTβR-Igwere analyzed in duplicate by nonreducing SDS-PAGE electrophoresis using4-20% gradient gels followed by western blotting.

FIG. 9: Effects of culture temperature on the percentage of deadmaterial in the total amount of human LTβR-Ig secreted from mammaliancells. Duplicate flasks containing CHO cells secreting recombinant humanLTβR-Ig were cultured at the indicated temperatures and the secretedhuman LTβR-Ig was purified by Protein A affinity chromatography. Thepercentage of the “inactive” form of human LTβR-Ig was assesses induplicate using analytical HIC chromatography. The X-axis represents theculture temperature, the Y-axis shows the amount of “inactive” materialexpressed as a percentage of the total amount of human LTβR-Ig secretedby the cells.

FIG. 10. Nonreducing SDS-PAGE analysis of HVEM-Ig prepared at 28, 32 and37° C.: Protein A purified preparations of HVEM-Ig derived from cellscultured at 28, 32, and 37° C. were mixed with nonreducing SDS-PAGEsample buffer and electrophoresed on precast 4-20% SDS-PAGE gels. Theprotein bands were visualized with coomassie blue. The band marked withan arrow represents unaggregated HVEM-Ig. The higher molecular weightbands visible on the gel correspond to aggregated forms.

FIG. 11: FACS analysis of the binding of HVEM-Ig generated at 37° vs 32°C. to surface Light and/or LTαβ. A). Binding of HVEM-Ig as a functionconcentration to 293 cells transfected with human LIGHT showning thatmaterial generated at 32 C binds better than 37 material. B). An exampleof the FACS profiles observed of HVEM-Ig binding at a concentration of 2ug/ml to LIGHT transfected 293 cells. C). A further example of theimproved binding of 32 C generated HVEM-Ig to the surface of the II-23 Tcell hybridoma line which displayes both LIGHT and surface lymphotoxinαβ(LTαβ) complex.

FIG. 12: A BIAcore analysis of the binding of either the LIGHT orlymphotoxin-α (LTα) ligands to BIA core chips immobilized HVEM-Iggenerated at three different temperatures. Each curve shows a bindingevent at one concentration of ligand and the following concentrationswere employed: 30, 15, 7.5, 3.75, 1.87, 0.93, 0.47, 0.23, 0.11 and 0.0ug/ml. Each chip was loaded to the same RU level indicating that equalamounts of receptor-Ig were bound.

DETAILED DESCRIPTION

Reference will now be made in detail to the claimed invention. Thisinvention concerns the ability to produce high levels of functional oractive forms of immunoglobulin fusion proteins of receptors in the TNFfamily. The success of clinical interventions with receptor-Ig fusionproteins requires a long term presence and the ability to treatchronically or a minimum during disease flares. Ideally, preparations ofsuch fusion proteins for human use will not have any aggregated,inactive or misfolded forms as their presence will reduce the potency ofthe drug and the altered structures could elicit antibody responses thatmay facilitate clearing of the drug thereby reducing its potency.Moreover, anti-receptor antibodies can directly cross link naturalreceptor on the cell surfaces thereby activating them, i.e. agonisticantibodies such as those described in Browning et al 1996, JEM.Agonistic antibodies activate the system and thus further receptor-Igtreatments may be less effective or even detrimental. By “immunoglobulinfusion proteins” we refer to any fusion of any functional portions ofthe extracellular domain of a polypeptide with any portion of theimmunoglobulin constant regions, e.g. the CH1, CH2 or CH3 domains orcombinations thereof. Preferably the polypeptide is a member of the TNFfamily of receptors. The portions of the Ig molecule may derive from anyof the various immunoglobulin isotypes, including, for example, IgG1,IgG2, IgM, IgA etc. By “TNF family of receptors” we refer to anyreceptor, whether naturally membrane bound or secreted (as in the caseof osteoprotegerin), which has the canonical TNF family cysteinebridging patterns or any receptor which binds to a defined member of theTNF family of ligands (e.g. Banner et al, 1993). The claimed inventionin other embodiments relates to TNF family receptor-Ig fusions obtainedby the methods discussed herein, as well as to pharmaceuticalpreparations comprising them.

LTβR-Ig protein is expressed in two forms in monkey cos cells or Chinesehamster ovary cells. One form binds ligand with high affinity, the“active” form whereas the other, the “inactive” form does not. Thismixture of live and dead forms has not been described previously for anyof the TNF receptor-Ig fusion proteins, however, the nature of theinactive form is not clear. The two forms were discovered by thepresence of two bands in an SDS-PAGE analysis. The expressed materialcontains considerable glycosylation heterogeneity; however, thisheterogeneity most likely does not result in the functional problemsdescribed here. For example, when the receptor is expressed as a solublemonomeric form lacking the transmembrane domain and the immunoglobulinFc region, non-, mono- and di-N-linked glycosylated forms are observed.Both single and double glycosylated forms can be immunoprecipitated byBDA8, an antibody that recognizes only functional forms. Moreover, theaffinity-purified forms have similar glycosylation heterogeneity. Morelikely, we speculate that the expression of a non-membrane anchored formleads to some aberrant disulfide linking resulting in inappropriatecrosslinking between the two arms of the receptor-Ig dimer.

The TNF family of receptors generally 3-4 repeated domains in theextracellular ligand binding portion with about 3 disulfide bridges perdomain. It is conceivable that inapproapriate folding would lead toeither an incorrect pattern of disulphide bonds or the lack of someformation of disulphide bonds (illustrated schematically in FIG. 2). Inthe case of a receptor-Ig fusion protein, it appears that the earlyfolding of the Fc domain enables its subsequent dimerization whichbrings the two LTBR chains, i.e. the two receptor arms, into closeproximity. If the receptor domains have not yet completed their folding,then there is the potential for free sulfhydryls to pair between thearms, i.e. interarm bridging. Such incorrect folding may occur, ordisulfide scrambling may result between free sulfhydryls juxtaposed nextto already formed disulfide linkages in both cases leading to foldingerrors. It is also possible that incorrect folding occurs within one armi.e. intra-arm folding errors, although such errors may not result in aradically different shape of the final molecule. In this case, activeand inactive forms may not be readily resolvable using conventionalsizing methods.

Lastly, in the TNFR55 receptor, the fourth domain, i.e. the domainclosest to the transmembrane region has been demonstrated to be criticalfor ligand binding when the receptor was expressed as an Ig fusionprotein (Marsters et al, 1992). This domain may be critical for many ofTNF receptors. Another study on TNFR55 showed that it was critical onlyin the context of the Ig fusion protein, and that the membrane formlacking the fourth domain was fully active in binding TNF ligand(Corcoran et al, 1994), However, more recently, crystallographicanalyses have pointed to possible critical functions associated with thefourth domain (Naismith et al, 1996). The fourth domain is relativelyconserved between species yet lacks direct contacts with the ligand(Banner et al, 1993). It is possible that interarm bridging in thisregion between the two fourth domains which lie next to the hinge andCH2+CH3 Fc domains would not appreciably alter the global shape of themolecule and hence may be invisible in size-based separation methods.Nonetheless, this molecule would exhibit impaired ligand binding.Receptors having only 3 domains could behave in a similar fashion.

Reducing the the temperature during cell culture resulted insignificantly less of the misfolded smaller form (i.e. the inactiveform) being secreted. This improvement is presumably due to a reducedfolding rate of the polypeptide which would allow for more time to foldthe individual domains of the LTβR portion prior to assembling thereceptor-Ig fusion protein into the expected dimer form. The absolutetemperature required to slow down the folding process is host dependent.For mammalian cells (i.e. CHO), the claimed method preferably occurs attemperatures of about 27° C. to about 35° C., more preferably, thetemperature is about 27° C. to about 32° C. Claimed methods also can beused in yeast culture systems. Yeast culture needs to be grown attemperatures of about 10° C. to about 25° C., preferably from about 15°C. to about 20° C. to achieve significant benefit.

Exploitation of the claimed invention allows the correct folding ofactive protein Ig fusions. It may be desirable, in some circumstances,to allow cell growth at higher temperatures, for example, about 37° C.to about 43° C., during which only a very low level of expression of thecloned gene will occur. After the desired growth period, the fusions canbe expressed at the low temperatures, to produce an increased yield ofactive fusions. The low temperatures in mammalian systems, as discussedabove, are preferably about 27° C. to about 35° C., more preferablyabout 27° C. to about 32° C.

Thus, the claimed methods, by lowering the temperature at which theprotein-Ig fusions are expressed, allow one skilled in the art toregulate the folding of both the protein and the Ig portions of thedesired protein.

Additionally, using the claimed method including affinity and/or theconventional chromatography techniques, one can now purify the activefractions sufficiently to use the chimeric proteins to blockimmunological function in various clinical settings. In addition, usingthe claimed methods having low temperature (i.e. 32° C. for CHO and ≦25°for yeast) cell culture conditions, it is now possible to prepareculture supernatant that is highly enriched in the larger, active formof human LTβR-Ig. Moreover, it is possible that other members of thisfamily of receptors suffer from similar problems. For example, we seetwo similar bands on non-reducing SDS PAGE for the TNFR55-Ig (alsocalled the p55 or p60 TNFR). Similarly, the properties of another TNFfamily receptor called HVEM are improved by secretion at lowertemperatures. This receptor may form an example of an intra-arm foldingerror as there are no obvious size differences in the materials made atthe various temperatures. Nonetheless, regardless of the mechanism, theclaimed methods having results in a higher percentage of loweredsecretion temperatures more active fusion proteins in preparations ofthe se and other member of the TNF family.

EXAMPLES Example 1 mAb that Specifically Recognize the Live Form ofHuman LTβR-Ig

The LTβR-Ig protein (FIG. 1) when secreted from either COS or CHO cellstransfected with a plasmid can be purified using standard protein Abased affinity chromatographic methods. The purified protein consists oftwo closely spaced bands at about 100 kDa on a nonreducing SDSacrylamide gel (FIG. 3). The two bands differ by about 5 kDa in apparentsize. When the protein is reduced, essentially two bands at about 50 kDaare resolved resulting from heterogeneous glycosylation (FIG. 3). Thetwo bands observed under nonreducing conditions, however, do not resultdirectly from the glycosylation differences that give rise to the pairof reduced bands. Using a panel of monoclonal antibodies to the humanLTβR, we showed that antibodies from group I, i.e. AGH1 and BDA8recognize only the large MW form of the receptor (Table I; all dataexcept the selectivity for the upper and lower bands was taken fromBrowning et al, 1996). Antibodies from this group bind directly to theligand binding region as evidenced by the observation that an Fabfragment of BDA8 can still block. Group II mAbs can also block ligandbinding, however, in biological assays, these mAbs show mixed agonistand antagonist behavior. When one makes an affinity column from thesemAbs (AGH1 was used in these experiments) and applies the mixture oflarge and small forms of the LTβR-Ig, the smaller form of the receptorflows through and the larger form sticks to the column matrix. Low pHelution yields a pure preparation of the large form (FIG. 4). The twofractions were assayed for their ability to bind to the surface phorbolester activated II-23 T cell hybridoma cells, i.e. cells expressingsurface lymphotoxin complex (as described in Browning et al, 1995), andonly the fraction that bound to the mAb was able to bind to surfacelymphotoxin. The flow through fraction, i.e. the lower MW band wascompletely inactive (FIG. 5). Specifically, supernatants from CHO cellsstably transfected with the LTβR-Ig construct were passed over a proteinA column to isolate the protein. Pure protein was eluted with 25 mMsodium phosphate buffer at pH 2.8 and the protein containing fractionswere neutralized with 1/10 volume of 0.5 M sodium phosphate, pH 8.6.Immunoprecipitations were carried out in a volume of 0.25 ml with 3 ugof LTβR-Ig and 4 ug of anti-LTβR mAb followed by capture of the mAb withKappaLock sepharose beads which recognize only the kappa chain on themouse mAb and not the human Fc domain. The beads were removed bycentrifugation and the supernatant was cleared of remainingimmunoglobulin with protein A sepharose. Beads were treated with SDSPAGE sample buffer (no reducing agent) and the buffer was loaded ontoSDS-PAGE gels. Gels were run and transferred onto Hybond and westernblotted with anti-human IgG Fc fragment conjugated to horse radishperoxidase (LTβR mAb followed by anti-mouse IgG-HRP andchemiluminescence detection of HRP (Amersham).

To exploit the ability of AGH1 that exclusively recognizes the activeform of LTβR:Ig, an AGH1 affinity column was prepared usingCNBr-activated sepharose (Pharmacia, Piscataway, N.J.) according tomanufacturers protocol. The column was extensively washed with PBS andthe protein A purified LTβR-Ig was applied and the flow throughcollected. The column was washed with PBS and then eluted with 25 mMsodium phosphate, pH 2. Fractions containing eluant were immediatelyneutralized as described above. Protein concentrations were determinedby absorbance at 280 nm assuming that a 1 OD solution equals 1 mg/ml.The flow-through and elution pools containing LTβR-Ig were tested forbinding by FACS analysis as described (Browning et al, 1995). Theflow-through fraction showed no FACS staining to cells expressingsurface LT whereas the elution pool retained full binding activity.

Example 2 Conventional Chromatographic Separation of Live and DeadComponents of Human LTβR-Ig

Potential structural differences between the large and small MWcomponents identified above are exploited in the design of separationmethods using conventional chromatography steps. As an example, theprotein can be sized by gel filtration in PBS to obtain partialseparation of the larger and smaller forms. These preparations can thenagain be applied to the same column to obtain preparations of the largerand smaller forms of human LTβR-Ig that are greater than 90% enriched inthe respective components. Alternatively, hydrophobic interactionchromatography (HIC) can be employed to achieve the same result. Theprotein mixture is diluted with ammonium sulfate, loaded on the HICcolumn and the large and small components are differentially eluted witha decreasing salt gradient. Under these conditions, baseline separationof the two human LTβR-Ig components is obtained (FIG. 6). These methodsas well as the imunoaffinity method described above are useful toprepare mg amounts of the large and small preparations of human LTβR-Igbut leave much to be desired for the preparation of large amounts ofthese components for pharmaceutical use. Applicants have invented a newmethod by adapting the HIC method to resins that can be obtained in bulkand have identified chromatography conditions that permit the small formof human LTβR-Ig material to flow through the column while the largecomponent is retained and can be selectively eluted (FIG. 6). The elutedmaterial can be subjected to a finishing step such as size exclusion orion exchange chromatography to remove aggregated material and otherimpurities and, which, after formulation into a suitable physiologicalbuffer can be used for in vivo work. The first example below (A)describes the specific conditions that were used to analytically assessthe amount of inactive material in a preparation of LTβR-Ig. The secondexample (B) describes the preprative purification process that resultsin a preparation of LTβR-Ig highly enriched in the active component.

A). Analytical Hydrophobic Interaction Chromatography (HIC) can be usedas a Quantitative Assay to Assess the Amounts of Inactive LTβR-Ig

Baseline resolution of the smaller, inactive and the larger, activecomponents of recombinant human LTβR-Ig was achieved on a PerseptiveBiosystems Poros ether/m column (4.6×100 mm, catalogue No. P091M526)equilibrated in 1.5 M ammonium sulfate and subsequent elution in adecreasing gradient of ammonium sulfate. The LTβR-Ig preparations werediluted to a concentration of 0.1 mg/ml and brought to a final buffercomposition of 1.5 M ammonium sulfate, 20 mM sodium phosphate, pH 9(buffer A). A portion (1 ml containing 100 μg of protein) was loadedonto the Poros ether/m column. The column was washed with 8.3 ml ofbuffer A. The active and inactive components were differentially elutedwith a linear gradient (total gradient volume of 16.6 ml) from 100%buffer A to 100% buffer B (20 mM sodium phosphate, pH 9) followed by a16.6 ml wash with buffer B. The column effluent was monitored forabsorbance at 214 nm. The whole procedure was carried out at ambienttemperature using a column flow rate of 1 ml/min. The elution profile ofa representative analytical HIC chromatogram is shown in FIG. 5. Peak 1contains the inactive fraction and peak 2 the active fraction ofLTβR-Ig. In order to quantify the relative contribution of the twoforms, the peak areas were integrated using the Perseptive instrumentsVison integration software.

B).Preparative Purification of Human Recombinant LTβR-Ig.

Clarification and concentration of conditioned media: The cell debriswas removed from 10L of conditioned media harvested from CHO cellssecreting recombinant LTβR-Ig using dead end filtration through a 5μpolypropylene 5 sqft. Calyx filter capsule (Microseparations Inc,Westborogh, Mass.) followed by a 0.2μ Opticap 4 inch filter cartridge(Millipore Corp., Bedford, Mass.). The clarified media was concentratedby ultra filtration to approximately 1 L using three S1Y30 SpiralUltrafiltration cartridges (Amicon, Beverly, Mass.) connected in series.

Protein A affinity Chromatography: The concentrated conditioned mediumwas passed by gravity through a 10 ml Protein A sepharose fast flow(Pharmacia) column at 4° C. The column was washed with 50 ml of PBS, 50ml of PBS containing 0.5 M NaCl, and 50 ml of PBS. To removecontaminating bovine IgG, the column was washed with 50 ml of 25 mMsodium phosphate, pH 5.5. The bound LTβR-Ig was eluted by gravity with25 mM sodium phosphate, 100 mM NaCl, pH 2.8 in 3 ml fractions andimmediately neutralized with 0.3 ml of 0.5 M sodium phosphate, pH 8.6.Fractions containing protein were identified by absorption spectroscopy,pooled and stored at −70° C.

Hydrophobic Interaction Chromatography: The protein A elution pool (40ml at a concentration of 2.5 mg/ml) was diluted with 40 ml of 3 M sodiumchloride, 40 mM sodium phosphate pH 7 and 20 ml of 1.5 M sodiumchloride, 20 mM sodium phosphate pH 7 (all solution were at ambienttemperature). The diluted pool was loaded onto a 10×100 mm (7.8 ml)Source PH15 (Pharmacia, Piscataway N.J.) at a flow rate of 2 ml/min. Thecolumn was washed with 79 ml of 1.5 M sodium chloride, 20 mM sodiumphosphate pH 7 at a flow rate of 20 ml/min. The bound protein was elutedwith 20 mM sodium phosphate pH 7 at a flow rate of 2 ml/min. Theabsorbance of the effluent was monitored at 280 nm and 9 ml fractionswere collected. Elution fractions containing protein were identified byUV absorption spectroscopy, pooled and stored at −70° C. FIG. 7represents a typical HIC elution profile. Under these conditions, theinactive material flows through the column and the active material isbound to the resin. The FIG. 7 insert contains a scan of the coomassieblue stained NR SDS-PAGE analysis of the follow-through and elutionpools, respectively.

Size Exclusion Chromatography: Approximately 100 ml (1.3 mg/ml) of theHIC elution pool containing the active components of LTβR-Ig wereconcentrated by ultrafiltration to 9 ml using a centriprep30concentrator (Amicon, Beverly, Mass.). The concentrate (10.3 mg/ml) wasloaded onto a 1.6×100 cm Superose-6 prep grade (Pharmacia, Uppsala,Sweden) column equilibrated in PBS at a flow rate of 1 ml/min. Theeffluent was collected in 3 ml fractions. Fractions containing proteinwere identified by UV absorption spectroscopy. Selected fractions wereanalyzed for aggregate content at a 3 μg/lane load using NR SDS-PAGE gelelectrophoresis. Fractions with minimal visible aggregate were pooledand stored frozen at −70° C.

In this fashion, LTβR-Ig can be prepared that contains minimal amountsof the inactive LTβR-Ig component that is present in the crude culturemedia.

Example 3 Low Temperature Fermentation Conditions Enrich for the Large,Active Component of Human LTβR-Ig During the Cell Culture Stage

Under conventional mammalian cell culture conditions, human LTβR-Ig issecreted as a mixture of approximately 50% small and 50% largecomponents. Using baculovirus infected insect cells to express the sameprotein results in drastically reduced levels of the small form. Asinsect cells are cultured at 28° C., we explored if human LTβR-Igsecreted from mammalian cells grown at low temperature would have analtered ratio of large and small forms. Shown in FIG. 8 are thesupernatants from CHO cells secreting human LTβR-Ig cultured in T-flasksat 28, 30, 33, 35, and 37° C. analyzed by western blot. The large andsmall forms (indicated by arrows) are present in the cultures grown at33, 35, and 37° C. Very little evidence of the small form can be seen inthe lanes containing culture supernatant from cells grown at 30 and 28°C. Thus, lowering the temperature during cell culture dramaticallyreduces the amount of the small, inactive form of human LTβR-Ig. Toquantify the relation ship between cell culture temperature and theextent of the enrichment for the larger, active form of human LTβR-Ig,duplicate culture flasks were set up and grown at temperatures rangingfrom 28-37° C. in one degree intervals. The protein A affinity purifiedhuman LTβR-Ig samples were analyzed by analytical HIC chromatography toquantify the ratio of the small and large components of human LTβR-Igpresent in the preparations derived from cells grown at the differenttemperatures. As shown graphically in FIG. 9, the amount of the lowerband rapidly decreases approximately 5 fold when the culture temperatureis lowered from 37 to 32° C. Lowering the culture temperature to 28° C.reduces the amount of the lower form but in a much less dramaticfashion. These results show that reducing the culture temperature byonly a few degrees from 37° C. dramatically decreases the amount of thesmaller mw component, thus increasing the yield of the larger activecomponent of human LTβR-Ig. Based on these data, culture temperatures of32 and 28° C. were selected to test if these observations could beduplicated on a large scale under conditions that would be suitable formanufacturing using CHO cells secreting recombinant human LTβR-Ig thathad been adapted to growth in suspension. For these experiments, thecells were grown to densities approaching 2×10⁶ cells/ml at 37° C. theculture was diluted with approximately 4 volumes of growth media andincubated at 32° C. until the cell viability dropped below 80%. Loweringthe temperature to 28° C. during the production phase also results insignificantly lower levels of the ìdeadî component in the final harvest.It was interesting to note that while the cell number did not increasevery much during the production phase at 28 or 32° C., a several foldincrease in product titer over culture grown exclusively at 37° C. wasobtained in the harvested conditioned media. The specific conditionsthat were used in the 32 and 28° C. process are described below.

Initial data suggests that lowering the culture temperature results insimilar benefits in other host systems such as yeast. It is interestingthat in yeast, the beneficial effects of low temperature production areobserved at much lower temperatures than in mammalian cells. Yeastcultured at 30° C. produce predominantly the inactive form, culturesgrown at 25° C. contain about an equal mixture of the inactive andactive forms and cultures fermented at 16° C. produce predominately theactive form of human LTβR-Ig. These observations suggest that lowtemperature fermentation will result in significantly higher yields ofthe active component of human LTβR-Ig in any secretory host system. Oneskilled in the art can easily determine the optimal productiontemperatures for each system.

Here we provide a detailed example of how this process was applied tothe production of LTβR-Ig. Two cell culture methods were developed thattake advantage of the fact that reducing the cell culture temperaturesignificantly reduces the amount of the inactive components present inLTβR-Ig secreted from host cells. An additional, unexpected benefit oflow temperature fermentation is a several fold improvement in titer whencompared to traditional fermentation runs carried out at 36-37° C. TableII summarizes the comparative yields and the relative amounts ofinactive LTβR-Ig components obtained with two different cell linestransfected with different LTβR-Ig constructs which varied in the extentof glycosylation (not germane to the thrust of this example).

32° C. Cell Culture Process: CHO cells secreting human LTβR-Ig that hadbeen adapted to growth in suspension were grown in DME/HAM's F-12 growthmedia (see Table III below) supplemented with 10% FBS, 140 mg/Lstreptomycin, and 50 mg/L gentamycin. For scale-up, two 750 ml-spinnerflasks were inoculated at approximately 2×10⁵ cells/ml in growth media.The cultures were grown at 37° C. in a 5% CO₂ atmosphere to a density ofapproximately 3×10⁶ cells/ml. The cell suspensions from both spinnercultures were combined to inoculate the scale-up bioreactor containingapproximately 10L of growth media. The culture was oxygenated at 11% O₂and grown for three days at 37° C. to a density of approximately 2×10⁶cells/ml. This culture was used to inoculate the production bioreactorcontaining 33L of growth medium at a density of 6.4×10⁵ cells/ml. Theproduction bioreactor was then cultured for 8 days at the beneficialtemperature of 32° C. with the oxygen sparge rate set at 11% O₂. Thecell density on the harvest day (day 8) was approximately 2×10⁶ cells/mlwith a viability of approximately 60%. Under these conditions, a titerof 12 mg/L LTβR-Ig was achieved which was two-fold higher than when thesame cells were cultured at the traditional temperature of 37° C. Therelative amount of the inactive component in the LTβR-Ig preparation was17% which represented a more than 60% decrease when compared to productobtained from a 37° C. culture.

28° C. Cell Culture Process: CHO cells secreting human LTβR-Ig that hadbeen adapted to growth in suspension were grown in DME/HAM's F-12 growthmedia (see table III below) supplemented with 10% FBS, 140 mg/Lstreptomycin, and 50 mg/L gentamycin. For scale-up, two 800 ml-spinnerflasks were inoculated at approximately 2×10⁵ cells/ml in growth media.The cultures were grown at 37° C. in a 5% CO₂ to a density ofapproximately 3.5×10⁶ cells/ml. The cell suspensions from both spinnercultures were combined to inoculate the scale-up bioreactor containingapproximately 10 L of growth media. The culture was oxygenated at 11% O₂and grown for two days at 37° C. to a density of approximately 1.7×10⁶cells/ml. This culture was used to inoculate two 40L and one 10 Lproduction bioreactor at starting cell densities of 2.5-3×10⁵ cells/mlusing a split ratio of 1:9. The production bioreactors were cultured at37° C. and oxygenated at 11% O₂until a cell density of approximately2×10⁶ cells/ml was reached (two days). At the end of day two, thebioreactor temperature was lowered to 28° C. and the bioreactors werecultured for an additional 5 days. On day 7, at cell densities ofapproximately 3×10⁶ cells/ml and cell viability of >75%, the bioreactorswere harvested and the conditioned media was processed as describedabove. Under these conditions, a final titer of approximately 20 mg/LLTβR-Ig was achieved which represents a 3.3 fold increase over the titerthat was obtained when the same cells are cultured at 37° C. Therelative proportion of the inactive component was 10% which represents a80% decrease over the material prepared at 37° C.

Example 4 Alterations in the Fc Domain to Minimize Dead Forms DuringProduction

Based on our hypothetical explanation of why dead molecules result inthese preparations, one would predict that slowing the time before Fcdomains would dimerize would increase the fraction of correctly foldedreceptor domains. We explored several methods to achieve this result. Itis possible that different Ig Fc domains would fold at different rates,yet exchange of the IgG1 domain for an IgG4 domain did not change thelive dead ratio. Secondly, the cysteine residues in hinge region thatcrosslink the two peptide chains were removed from the IgG1 Fc domain bymutagenesis. The Fc domains can dimerize well in the absence of hingedisulfide formation but the rate may be slower in its absence.Replacement of the two cysteine residues by alanine resulted in adecreased amount of dead form as quantitated by SDS-PAGE and HICchromatography such that where wild type LTβR-Ig would contain 50 and 5%dead forms at 37 and 28° C., the deletion of the cysteines from the IgG1hinge lead to 20 and 5% dead form when produced at these respectivetempertures.

Therefore, hinge modification by replacement of both cys residues canimprove the quality of a preparation and, moreover, it is possible thatreplacement of only one cys residue could have a beneficial effect. Suchgenetic modifications could reduce the percentage of dead form withrecourse to low temperature production methods.

Example 5 Deletion of Cystine Bridges to Correct Folding Problems

Typically, it is very difficult to define the folding pathways utilizedby a protein to get to the final correct form. Nonetheless, somedisulfide bridges in the TNF receptor family are unusual and may not beneeded for the final folded state. These bridges are good candidates formutageneisis. One such bridge in the third domain of LTβR-Ig was removedby conventional site directed mutagenesis of cysteines 101 and 108 toalanines (using the numbering from the sequence defined in Ware et al,1995) led to an improved ratio of dead/live material as evidenced bySDS-PAGE. With wild type LTβR-Ig typically shows the presence of 50 and5% dead form when produced at 37 and 28° C. respectively. The mutantform with the one cystein disulfide bridge deleted had 20 and 5% deadform when produced at these temperatures.

Example 6 Use of Lower Temperatures to Improve the Quality of aPreparation of HVEM-Ig

Herpes virus entry mediator (HVEM) is a TNF family receptor related tothe LTβR and binds tightly to the ligand LIGHT tightly and weakly to theligand lymphotoxin-a (LTa) (Mauri et al, 1998). Human HVEM was preparedas a Ig fusion protein by PCR amplification of the extracellular domainand fused to the human IgG1 CH2 and CH3 region as described for LTβR-Ig(Crowe et al, 1994). The construct was inserted into a vector calledCH269 (Chicheportiche et al, 1997) for transient expression in the humanembryonic kidney cell line 293 with high copy vector expression usingthe EBNA system (293-E cells). Supernatents were collected and HVEM-Igpurified using ProteinA affinity chromatography and low pH elution.Recombinant LTα was prepared from insect cells as described (Browning etal, 1996a). Recombinant soluble human LIGHT was prepared by PCRamplification of the entire cDNA using RNA from activated II23 cellsyielding the coding region of the sequence described by Mauri et al,1998. The receptor binding domain of LIGHT was amplified by PCR andfused onto the alpha mating factor leader sequence and expressedessentially as described for other related proteins (Browning et al,1996). A FLAG tag and (G₄S)₃ spacer amino acid sequences were insertedbetween the leader and the receptor binding domain such that thesecreted LIGHT would possess a N-terminal FLAG sequence. The constructencoded the following molecule:

“MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR..EADYKDDDDNGGGSGGGSGGGSKELNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERL VRLRDGTRSYFGAFMV”where the two dots indicates the expected N-terminus of the matureprotein which could be further processed by removal of the next twoamino acids (EA). The protein was purified from the supernatant byaffinity chormatography over an anti-FLAG mAb column and elution witheither low pH or calcium chelation. Full length LIGHT was also insertedinto a vector CH269 for expression on the surface of 293-E cells asdescribed (Chicheportiche et al, 1997). FACS binding methods for thedetection of receptor-Ig to cell surfaces and BIAcore methods formeasuring the binding of soluble ligands to immobilized receptor-Ig havebeen described (Mackay et al, 1997). The BIAcore technology yields realtime measurements of protein bound to the chips (i.e.

the receptor).

CHO cells expressing HVEM-Ig were grown to confluency at 37 C in rollerbottles using growth media supplemented with 10% FBS. When the cellsreached confluence, the spent media was exchanged with fresh growthmedia and the cultures were incubetaed at 37, 32 and 28 C. The 28 and 32C cultures were harvested once a week, the 37 C culture every 4 days.The secreted HVEM-Ig was purified using Protein A affinitychromatography as described. The purified preparations were stored at−70 C until analyses. When HVEM-Ig was produced at 28, 32 and 37° C. andpurified, all the preparations behaved similarity on SDS-PAGE analysis(FIG. 10) suggesting that large alterations in the protein did notoccur. Therefore, if aberrant folding/disulfide bridging occurred, itwas either intra-arm or occurred close to the hinge region of the Fcdomain, i.e. inter-arm bridging and hence did not appreciably affect theoverall shape of the molecule in SDS-PAGE. The ability of thereceptor-Ig to bind to LIGHT expressed on 293-E cells or LIGHT onactivated II23 cells was assessed in FACS binding assays (FIG. 11). Onboth cell types, the ability of HVEM-Ig to bind cell surface ligand wasimproved roughly 2-3 fold upon expression at 32 C. Using BIAcoretechnology, it was observed that when BIAcore chips were loaded withHVEM-Ig to similar levels (RU values reflect amount of protein on thechip), HVEM-Ig produced at lower temperatures bound more ligand thanproteins produced at the higher temperatures (FIG. 12). This result wasobtained whether LIGHT or LTα was the ligand. The BIAcore binding curvesshow the realtime on and off binding events and it can be seen that thebinding events were similar regardless of the production temperature.Therefore, a portion of the preparation is effectively dead and thisproportion is minimized by lower production temperatures. We speculatethat the lower temperature has corrected an abberant folding problem andimproved the percentage of live molecules even though we cannot directlyobserve the fraction of dead molecules. Affinity chromographictechniques as outlined above could serve to resolve the live/dead formsfollowing optimization of the preparation by lowered growth temperaturesand or mutagenesis of various cysteines either in the hinge or in thereceptor itself to prevent incorrect folding.

Example 7 Generic Schemes to Minimize Dead Forms of Other TNF-FamilyReceptor-Ig Fusion Proteins

Most receptors of the TNF family have been prepared as fusion proteinconstructs with immunoglobulin-Fc domains:

Reference p55 TNF-R (Loestcher et al, 1991, Marsters et al, 1992;Ashkenazi et al, 1991) p75 TNF-R (Mohler et al, 1993) LTβ-R (Crowe etal, 1994) Fas (Suda et al, 1993) CD27 (Goodwin et al, 1993) CD30 (Smithet al, 1993) CD40 (Fanslow et al, 1992) Ox40 (Baum et al, 1994) 4-1BB(Alderson et al, 1994) HVEM (Mauri et al, 1998)In several cases, most notably, Fas-Ig and CD40-Ig e.g. Fanslow et al,1992, the chimeras are poorly active relative to the soluble Fc forms ofthe two TNF receptors. It is possible that some of these preparationsare mixtures of live and dead forms in varying ratios. A dead formrefers simply to a molecule that binds with a affinity substantially(10-1000 fold) lower than the live form, i.e. it may not be completelylack binding activity, but instead has a reduced affinity for ligandrelative to the high affinity form found on cells naturally. Abberantinter-receptor arm or intra-receptor arm disulphide linkages may occurleading to a less active protein.

With any of these receptors or other as yet undefined receptors, a panelof anti-receptor mAbs would be prepared by conventional technologies.Those antibodies able to block the binding of the ligand to the receptorpreferably as assessed using a ligand binding assay to the nativereceptor on a cell surface (although other methods using recombinantreceptor forms may suffice) would be used to form affinity columns. Thepreparation of the mixture of live and dead receptor-Fc forms would bepassed over the column and the flow through collected. The material thatbound to the column would be eluted with a low pH buffer (typically pH2.5 to 4.0) immediately neutralized. The two fractions would be bound toeither cells in a FACS binding assay (or any other standard bindingformat) or ligand at varying protein concentrations. Some of these mAbswill selectively bind to the live form and a difference between theconcentration needed to get 50% binding of the flow through vs eluatewill be seen. This result marks that mAb as a demarcating mAb. The ratioof the protein in the eluate to the flow through will indicate thepercent live form in the preparation.

These mAbs thus identified can be used to affinity purify the live form.Moreover, their use in various immunoassay formats can be used tooptimize for expression of the correct form of the desired receptor-Fcform. The assay can further be used in conjunction with otherconventional purification methods to find methods that would purify theactive form of the receptor without resort to affinity techniques. Usingthese Abs to delineate live and dead forms, the culture temperaturecould be optimized and chromatographic methods developed to enrich forthe live form. Alternatively, HIC column methods could be exploited toseparate live and dead forms and using this method, culture conditionscould be optimized. Likewise, these assays would form the basis forcysteine mutagenesis of the receptor portion to define problem disulfidebonds which upon removal would yield functionally active material.

As many of these receptors will have application as therapeutic agentsin human disease and one will want to put only properly folded formsinto a patient, these methods for both defining the preparation andremoving poor binding forms will have utility.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods of the presentinvention without departing from the spirit or scope of the invention.Thus, it is intended that the present invention cover the modificationsand variations of this invention provided that they come within thescope of the appended claims and their equivalents.

TABLE I Summary of Anti-LT-b-R mAbs HT29 Cytotoxicity Blocking mAbSoluble Soluble Receptor mAb cell Receptor Immobilized mAb mAb with BandmAb Name Staining Binding^(a) on Plastic^(b) alone LTa1/b2Precipitated^(f) Group^(g) BDA8 +++ +++ ++ +/− −−−^(c) upper I AGH1 ++++++ +/− −−− upper I BCG6 +++ ++ +++ +/− +/− both II BHA10 +++ ++++/−^(e) +/− +/− nd II BKA11 +++ +/− +++ − +++^(d) both III CDH10 +++ ++++/− +++ nd III CBE11 +++ nd +++ +/− − both IV ^(a)Assay assessed whetherantibody blocks binding of soluble receptor to activated II-23. nd = notdone. ^(b)Goat anti-mouse Fc coated plate, captured anti-receptor mAb,HT29s plus IFNg. ^(c)Blocks ^(d)Potentiates ^(e)Variable, some partialinhibition in some assays, none in others. ^(f)Receptor formprecipitated using mAb plus KappaLock system. ^(g)Groups were defined onthe basis of the data in this table plus epitope mapping done usingBIAcore technology.

TABLE II Expression of LTβR-Ig constructs in CHO cells cultured atdifferent culture temperatures. Fermentation Expression % InactiveConstruct Temperature ° C. mg/L^(a) Components^(b) LTβR05 37  6 50 32 1217 28 20 10 LTβR09 37 10 50 32 — — 28 76  6 ^(a)The expression level wasassessed using Protein A affinity chromatography. ^(b)The amount ofinactive components present in the LTβR-Ig preparation was assessedafter Protein A affinity purification using the analytical HIC method.

TABLE III DME/HAM's F-12 Growth Media Supplements Component Amount in 1L growth media^(a) Fetal Bovine Serum 100 ml Glucose 1.85 g Ammoniumbicarbonate 2.2 g Streptomycin 140 mg Gentamycin 50 mg Ethanolamine (1Mstock) 0.1 ml Lipoic acid 91.2 mg Linoleic acid 38.4 mgTriiodo-L-Thyronine 0.2 mg Ex-Cyte VLE (Bayer) 1 ml Bovine Insulin 10 mgBovine Transferrin 10 mg Bovine Serum Albumin 50 mg Pluronic F-68 1 gCysteine 82 mg Methionine 34 mg Serine 52 mg Valine 105.6 mg Glycine 50mg Aspartic Acid 24.4 mg Proline 52.2 mg ^(a)Components are mixed withthe base media powder and the volume is then brought to 1 L. The pH ofthe media is adjusted to 7.20 ñ 7.25 using 50% HCl.

REFERENCES

-   Aggarwal, B. and Natarajan, K. (1996) Eur. Cytokine Rev. 7:93-   Alderson, M. R. et al. (1994) Eur J Immunol 24, 2219-27-   Ashkenazi, A. et al. (1991) Proc Natl Acad Sci USA 88, 10535-9-   Banner, D. W. et al (1993) Cell 73, 431-445-   Baum, P. R. et al. (1994) Embo J 13, 3992-4001-   Bazzoni, F. and Beutler, B. (1996) New England J. Med. 334:1717.-   Browning, J. L. et al. (1995) J. Immunol. 154, 33-46-   Browning, J. et al, (1996) J. Exp. Med. 183, 867-878-   Browning, J.L. et al. (1996a) J. Biol. Chem. 271, 8618-8628-   Bucay, N. et al. (1998) Genes and Development 12:1260-   Chaplin, D. and Fu, Y-X. (1998) Current Opinion in Immunology 10,    289-297-   Chiceportiche, Y. et al (1997) J. Biol. Chem. 272, 32401-32410-   Corcoran, A. et al (1994) Eur. J. Biochem. 223, 831-840-   Crowe, P. D. et al. (1994) Science 264, 707-10-   Eason, J. D. et al. (1996) Transplantation 61:224.-   Eggermont, A. M. et al. (1996) J. Clin. Oncology 14:2653-   Fanslow, W. C. et al. (1992) J Immunol 149, 655-60-   Feldmann, M. et al. (1997) An. Immunol. 64:283-350.-   Goodwin, R. G. et al. (1993) Cell 73, 447-56-   Green, D. and Ware, C. F. (1997) Proc. Natl. Acad. Sci USA 94:5986-   Harrop, J.A., et al, (1998) J. Biol. Chem. 273:27548.-   Loetscher, H. et al. (1991) J Biol Chem 266, 18324-9-   Mackay, F. et al. (1998) Gastroenterology 115: 1484-1475-   Mackay, F and Browning, J. (1998) Nature 395:26-   Mackay, F. et al. (1997) Eur J Immunol 27, 2033-42-   Marsters, S. et al (1992) J. Biol. Chem. 267, 5747-5750-   Mauri, D. N. et al. (1998) Immunity 8, 21-30-   Mohler, et al. (1993) J Immunol 151, 1548-61-   Naismith, J. et al (1996) J. Mol. Recognition 9, 113-117-   Rennert, P. D. et al. (1996) J Exp Med 184, 1999-2006-   Rennert, P. D., Browning, J. L., and Hochman, P. S. (1997) Int    Immunol 9, 1627-39-   Simmonet, W. S. et al. (1997) Cell 89:309.-   Smith, C. A., Farrah, T., and Goodwin, R. G. (1994) Cell 76, 959-62-   Smith, C. A. et al. (1993) Cell 73, 1349-60-   Suda, T., Takahashi, T., Golstein, P., and Nagata, S. (1993) Cell    75, 1169-78-   Van Dullemen, H. M. et al. (1995) Gastroenterology 109:129-   Ware, C. F. et al. (1995) Current topics in Microbiology and    Immunology 198, 175-218

1-36. (canceled)
 37. A method for increasing expression of active tumornecrosis factor (TNF) receptor family member-Ig Fc fusion proteins andminimizing expression of inactive TNF receptor family member-Ig Fcfusion proteins comprising culturing a mammalian host cell transformedwith DNA molecule encoding a desired TNF receptor -Ig fusion protein ina mammalian cell culture having a minimum temperature of about 27° C. toa maximum temperature of less than or equal to 30° C., wherein thetransformed host cell is first cultured at a temperature of about 33° C.to about 37° C. for a period of time sufficient to allow growth of saidhost cell, and wherein the active TNF receptor family member is involvedin immune regulation and comprises 2-4 copies of canonical TNF familycysteine-rich domains.
 38. The method of claim 37, wherein the TNFreceptor family member is a lymphotoxin-β receptor, TNFR-55, TNFR-75,HVEM or a ligand-binding portion thereof.
 39. The method of claim 38,wherein the TNF receptor family member is a lymphotoxin-β receptor or aligand-binding portion thereof.
 40. The method of any one of claims37-39, further comprising the step of recovering the active TNF receptorfamily member-Ig Fc fusion proteins from the culture.
 41. The method ofclaim 40, wherein the active TNF receptor family members are recoveredby hydrophobic interaction chromatography.
 42. The method of claim 40,wherein the active TNF receptor family member-Ig Fc fusion proteins arerecovered based on their ability to bind an antibody that binds directlyto the ligand binding region of the TNF receptor family member andexclusively recognizes the active form of the TNF receptor familymember-Ig Fc fusion protein.
 43. The method of claim 37, whereinthe_mammalian host cell is a CHO cell.
 44. The method of claim 37,wherein the Ig Fc region is human.
 45. The method of claim 37, whereinthe Ig Fc region is of an IgG1 isotype.